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Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

ABSTRACT: The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase-encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole-mount in situ hybridization protocols to detect gene expression in the anatomical three-dimensional context of whole juvenile brains. To confirm whether th2-expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2-expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish.

SUBMITTER: Filippi A

PROVIDER: S-EPMC2841823 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

Filippi Alida A Mahler Julia J Schweitzer Jörn J Driever Wolfgang W

The Journal of comparative neurology 20100201 4

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication a ...[more]

PMID: 20017209

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Project description:The homeodomain transcription factor Orthopedia (Otp) is an important regulator for specification of defined subsets of neuroendocrine cells and dopaminergic neurons in vertebrates. In zebrafish, two paralogous otp genes, otpa and otpb, are present in the genome. Neither complete loss of Otp activity nor differential contributions of Otpa and Otpb to specification of defined neuronal populations have been analyzed in detail. We characterized zebrafish embryos and early larvae mutant for null alleles of otpa, otpb, or both genes to determine their individual contributions to the specification of th expressing dopaminergic neuronal populations as well as of crh, oxt, avp, trh or sst1.1 expressing neuroendocrine cells. otpa mutant larvae show an almost complete reduction of ventral diencephalic dopaminergic neurons, as reported previously. A small reduction in the number of trh cells in the preoptic region is detectable in otpa mutants, but no significant loss of crh, oxt and avp preoptic neuroendocrine cells. otpb single mutant larvae do not display a reduction in dopaminergic neurons or neuroendocrine cells in the otp expressing regions. In contrast, in otpa and otpb double mutant larvae specific groups of dopaminergic neurons as well as of crh, oxt, avp, trh and sst1.1-expressing neuroendocrine cells are completely lost. These observations suggest that the requirement for otpa and otpb function during development of the larval diencephalon is partially redundant. During evolutionary diversification of the paralogous otp genes, otpa maintained the prominent role in ventral diencephalic dopaminergic and neuroendocrine cell specification and is capable of partially compensating otpb loss of function. In addition, we identified a role of Otp in the development of a domain of somatostatin1-expressing cells in the rostral hindbrain, a region with strong otp expression but so far uncharacterized Otp function. Otp may thus be crucial for defined neuronal cell types also in the hindbrain.

Project description:BACKGROUND: Dopaminergic neurons form in diverse areas of the vertebrate di- and mesencephalon to constitute several major neuromodulatory systems. While much is known about mammalian mesencephalic dopaminergic neuron development, little is known about the specification of the diencephalic dopaminergic groups. The transcription factors Pitx3 and Lmx1b play an important role in mammalian mesencephalic dopaminergic specification, and Nurr1/Nr4a2 has been shown to contribute to specification of the dopaminergic neurotransmitter phenotype. We use zebrafish to analyze potentially evolutionarily conserved roles of these transcription factors in a vertebrate brain that lacks a mesencephalic dopaminergic system, but has an ascending dopaminergic system in the ventral diencephalon. RESULTS: We use a combination of fluorescent in situ hybridization and immunohistochemistry to determine whether nr4a2, lmx1b, and pitx3 genes are expressed in mature dopaminergic neurons or in potential precursor populations. We identify a second nr4a2 paralogue, nr4a2a, and find it co-expressed with Tyrosine hydroxylase in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2b is only expressed in preoptic and retinal dopaminergic neurons. Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down of both nr4a2a and nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for tyrosine hydroxylase and dopamine transporter expression. Zebrafish lmx1b.1 is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down reveals that it is specifically required for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons. Both lmx1b genes and pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that might contain precursor cells for ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmx1b paralogues, the number of neurons in diencephalic dopaminergic clusters with ascending projections appears specifically reduced. Thus lmx1b paralogues may contribute to the generation of diencephalic dopaminergic precursors. Conversely, knock-down of pitx3 does not specifically affect any diencephalic DA cluster. CONCLUSION: Our data indicate a conserved evolutionary role of Nr4a2 proteins in specification of the neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. For zebrafish lmx1b genes, which are not expressed in mature dopaminergic neurons, our data suggest a role in diencephalic precursor populations contributing to the ascending dopaminergic systems. A di-mesencephalic longitudinal domain of lmx1b expression may be the basis for the expansion and posterior shift of ventral di-/mesencephalic dopaminergic populations with ascending projections during evolution.

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Dataset Information

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

Publications

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

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